Thomas Heitzer

Increased NADH-Oxidase–Mediated Superoxide Production in the Early Stages of Atherosclerosis Evidence for Involvement of the Renin-Angiotensin System

BACKGROUND Angiotensin II activates NAD(P)H-dependent oxidases via AT1-receptor stimulation, the most important vascular source of superoxide (O2*-). The AT1 receptor is upregulated in vitro by low-density lipoprotein. The present study was designed to test whether hypercholesterolemia is associated with increased NAD(P)H-dependent vascular O2*- production and whether AT1-receptor blockade may inhibit this oxidase and in parallel improve endothelial dysfunction. METHODS AND RESULTS Vascular responses were determined by isometric tension studies, and relative rates of vascular O2*- production were determined by use of chemiluminescence with lucigenin, a cypridina luciferin analogue, and electron spin resonance studies. AT1-receptor mRNA was quantified by Northern analysis, and AT1-receptor density was measured by radioligand binding assays. Hypercholesterolemia was associated with impaired endothelium-dependent vasodilation and increased O2*- production in intact vessels. In vessel homogenates, we found a significant activation of NADH-driven O2*- production in both models of hyperlipidemia. Treatment of cholesterol-fed animals with the AT1-receptor antagonist Bay 10-6734 improved endothelial dysfunction, normalized vascular O2*- and NADH-oxidase activity, decreased macrophage infiltration, and reduced early plaque formation. In the setting of hypercholesterolemia, the aortic AT1 receptor mRNA was upregulated to 166+/-11%, accompanied by a comparable increase in AT1-receptor density. CONCLUSIONS Hypercholesterolemia is associated with AT1-receptor upregulation, endothelial dysfunction, and increased NADH-dependent vascular O2*- production. The improvement of endothelial dysfunction, inhibition of the oxidase, and reduction of early plaque formation by an AT1-receptor antagonist suggests a crucial role of angiotensin II-mediated O2*- production in the early stage of atherosclerosis.

Antioxidant Vitamin C Improves Endothelial Dysfunction in Chronic Smokers

BACKGROUND Chronic smoking is associated with endothelial dysfunction, an early stage of atherosclerosis. It has been suggested that endothelial dysfunction may be a consequence of enhanced degradation of nitric oxide secondary to formation of oxygen-derived free radicals. To test this hypothesis, we investigated the effects of the antioxidant vitamin C on endothelium-dependent responses in chronic smokers. METHODS AND RESULTS Forearm blood flow responses to the endothelium-dependent vasodilator acetylcholine (7.5, 15, 30, and 60 micrograms/min) and the endothelium-independent vasodilator sodium nitroprusside (1, 3, and 10 micrograms/min) were measured by venous occlusion plethysmography in 10 control subjects and 10 chronic smokers. Drugs were infused into the brachial artery, and forearm blood flow was measured for each drug before and during concomitant intra-arterial infusion of the antioxidant vitamin C (18 mg/min). In control subjects, vitamin C had no effect on forearm blood flow in response to acetylcholine and sodium nitroprusside. In contrast, in chronic smokers the attenuated forearm blood flow responses to acetylcholine were markedly improved by concomitant administration of vitamin C, whereas the vasodilator responses to sodium nitroprusside were not affected. CONCLUSIONS The present studies demonstrate that the antioxidant vitamin C markedly improves endothelium-dependent responses in chronic smokers. This observation supports the concept that endothelial dysfunction in chronic smokers is at least in part mediated by enhanced formation of oxygen-derived free radicals.

Heparins Increase Endothelial Nitric Oxide Bioavailability by Liberating Vessel-Immobilized Myeloperoxidase

Background— Neutrophils and monocytes are centrally linked to vascular inflammatory disease, and leukocyte-derived myeloperoxidase (MPO) has emerged as an important mechanistic participant in impaired vasomotor function. MPO binds to and transcytoses endothelial cells in a glycosaminoglycan-dependent manner, and MPO binding to the vessel wall is a prerequisite for MPO-dependent oxidation of endothelium-derived nitric oxide (NO) and impairment of endothelial function in animal models. In the present study, we investigated whether heparin mobilizes MPO from vascular compartments in humans and defined whether this translates into increased vascular NO bioavailability and function. Methods and Results— Plasma MPO levels before and after heparin administration were assessed by ELISA in 109 patients undergoing coronary angiography. Whereas baseline plasma MPO levels did not differ between patients with or without angiographically detectable coronary artery disease (CAD), the increase in MPO plasma content on bolus heparin administration was higher in patients with CAD (P=0.01). Heparin treatment also improved endothelial NO bioavailability, as evidenced by flow-mediated dilation (P<0.01) and by acetylcholine-induced changes in forearm blood flow (P<0.01). The extent of heparin-induced MPO release was correlated with improvement in endothelial function (r=0.69, P<0.01). Moreover, and consistent with this tenet, ex vivo heparin treatment of extracellular matrix proteins, cultured endothelial cells, and saphenous vein graft specimens from CAD patients decreased MPO burden. Conclusions— Mobilization of vessel-associated MPO may represent an important mechanism by which heparins exert antiinflammatory effects and increase vascular NO bioavailability. These data add to the growing body of evidence for a causal role of MPO in compromised vascular NO signaling in humans.

Systemic Endothelial Dysfunction as an Early Predictor of Adverse Outcome in Heart Failure

Objective—Endothelial dysfunction is an early event in the natural progression of heart failure. Increased oxidative stress has been linked to impaired endothelial function and both may play a prognostic role. Methods and Results—Endothelium-dependent and endothelium-independent vasodilatation were determined in 289 patients with mild left ventricular dysfunction by measuring forearm blood flow responses to acetylcholine and sodium nitroprusside using venous occlusion plethysmography. Vascular effects of the coadministration of the antioxidant vitamin C at pharmacological doses (24 mg/min) were assessed. Occurrence of death, heart transplantation, and readmission with worsening heart failure were recorded as clinical outcome parameters during a follow-up period of 4.8 years. Patients experiencing adverse events (n=79) had lower vasodilator responses to acetylcholine (P<0.001) and to sodium nitroprusside (P=0.03) compared with patients without events. However, beneficial effects of vitamin C did not differ between both groups. Cox proportional hazards model demonstrated that age (P=0.001), renal function (P=0.001), and blunted acetylcholine-induced vasodilatation (P=0.007) remained independent predictors of adverse outcome. Conclusions—Impaired peripheral endothelial function independently predicts long-term adverse outcome in patients with early-stage heart failure. The findings suggest that assessment of peripheral endothelial function may represent an additional mean for risk stratification and therapy management in these patients.

Beneficial effects of α-lipoic acid and ascorbic acid on endothelium-dependent, nitric oxide-mediated vasodilation in diabetic patients: relation to parameters of oxidative stress

The impairment of nitric oxide (NO)-mediated vasodilation in diabetes has been attributed to increased vascular oxidative stress. Lipoic acid has been shown to have substantial antioxidative properties. The aim of this study was to assess the effect of lipoic acid on NO-mediated vasodilation in diabetic patients in comparison with the well-recognized effect of ascorbic acid. Using venous occlusion plethysmography, we examined the effects of lipoic acid (0.2 mM) and ascorbic acid (1 and 10 mM) on forearm blood flow responses to acetylcholine, sodium nitroprusside and concomitant infusion of the NO-inhibitor, N(G)-monomethyl-L-arginine, in 39 diabetic patients and 11 control subjects. Plasma levels of antioxidants and parameters of lipid peroxidation were measured and correlated to endothelial function tests. Lipoic acid improved NO-mediated vasodilation in diabetic patients, but not in controls. NO-mediated vasodilation was improved by ascorbic acid at 10 mM, but not 1 mM. Improvements of endothelial function by ascorbic acid and lipoic acid were closely related. The beneficial effects of lipoic acid were positively related to plasma levels of malondialdehyde and inversely related to levels of ubiquinol-10. These findings support the concept that oxidative stress contributes to endothelial dysfunction and suggest a therapeutic potential of lipoic acid particularly in patients with imbalance between increased oxidative stress and depleted antioxidant defense.

The physiology and pathophysiology of the nitric oxide/superoxide system

SummaryThe endothelium modulates vascular tone by producing vasodilator vasoconstrictor substances. Of these, the most well characterized and potentially important are NO and.O2−. These small molecules exhibit opposing effects on vascular tone, and chemically react with each other in a fashion which negates their individual effects and leads to the production of potentially toxic substances. These dynamic interactions may likely have important implications, altering not only tissue perfusion but also contributing to the process of atherosclerosis..NO is produced in endothelial cells by an enzyme termed nitric oxide synthase. The endothelial.NO-synthase is activated when the intracellular level of calcium is increased. This occurs in response to neurohormonal stimuli and in response to shear stress. Acetylcholine and substance P are examples of neurohumoral substances that are able to stimulate the release of nitric oxide and to assess endothelial regulation of vasomotor tone. Importantly, the vasodilator potency of nitric oxide released by the endothelium is abnormal in a variety of diseased states such as hypercholesterolemia, atherosclerosis and diabetes mellitus. This may be secondary to decreased synthesis of nitric oxide or increased degradation of nitric oxide due to superoxide anions. More recent experimental observations demonstrate increased production of superoxide in atherosclerosis, diabetes mellitus and high renin hypertension suggesting that endothelial dysfunction in these states is rather secondary to increased.NO metabolism rather than due to decreased synthesis of.NO. Superoxide rapidly reacts with nitric oxide to form the highly reactive intermediate peroxynitrite (ONOO−). Peroxynitrite can be protonated to form peroxynitrous acid which in turn can yield the hydroxyl radical (OH.). These reactive species can oxidize lipids, damage cell membranes, and oxidize thiol groups..NO given locally, exerts potent antiatherosclerotic effects such as inhibition of platelet aggregation, inhibition of adhesion of leukocytes and the expression of leukocyte adhesion molecules. It is important to note, however, that in-vivo treatment with.NO (via organic nitrates) increases rather than decreases oxidant load within endothelial cells. It remains therefore questionable whether systemic treatment with.NO may have antiatherosclerotic properties or whether.NO may initiate or even accelerate the atherosclerotic process.ZusammenfassungDas Endothel reguliert durch die Produktion vasodilatierender und vaskonstriktorisch wirkender Substanzen in entscheidendem Maße den Gefäßtonus. Zu den am besten charakterisierten Molekülen gehören Stickstoffmonoxid (.NO) und das Superoxidanion (.O2−)..NO wird im Endothel durch das Enzym.NO-Synthase aus der Aminosäure L-Arginin gebildet. Die wichtigsten physiologischen Stimuli zur.NO-Bildung sind die pulsatile Dehnung der Gefäßwand sowie die Scherkräfte, die auf das Endothel einwirken. Pharmakologisch kann die Freisetzung von.NO in vitro und in vivo durch die Gabe von Acetylcholin induziert werden, ein Verfahren, das mit Erfolg zur Endothelfunktionsdiagnostik in peripheren Arterien und Koronararterien eingesetzt wird. Die vasodilatierende Wirkung von.NO ist im Rahmen von verschiedenen Erkrankungen wie arterielle Hypertonie, Hypercholesterinämie und Diabetes mellitus deutlich abgeschwächt. Dies kann jedoch nicht auf eine verminderte.NO-Bildung zurückgeführt werden, da die Aktivität bzw. die Expression des Enzyms endotheliale.NO-Synthase bei diesen Erkrankungen eher kompensatorisch gesteigert als abgeschwächt ist. Neuere tierexperimentelle Untersuchungen konnten nachweisen, daß bei diesen Krankheitsbildern die endotheliale Superoxidproduktion ebenfalls angekurbelt ist. Somit kommt einem erhöhten.NO-Metabolismus durch.O2− reagiert rasch mit.NO unter der Bildung des instabilen Metaboliten Peroxynitrit (ONOO−). Durch eine Protonierungsreaktion kann letztendlich das Hydroxylradikal entstehen, das wiederum Lipide und Thiolgruppen oxidieren und Membranen schädigen kann. In vitro konnten für.NO vielschichtige antiarteriosklerotische Eigenschaften. wie zum Beispiel Inhibition der Expression von Leukozytenadhäsionsmolekülen und Inhibition der Plättchenaggregation, nachgewiesen werden. In-vivo-Behandlung mit.NO (zum Beispiel in Form von Nitroglycerin) führt in Tiermodellen nachweislich eher zu einer Steigerung als zu einer Reduktion der endothelialen Superoxidbildung. Somit bleibt abzuwarten, ob durch eine Therapie mit.NO der bei Patienten mit manifester koronarer Herzkrankheit (und somit erhöhtem oxidativen Streß in der Gefäßwand) die Arteriosklerose positiv beeinflußt wird oder ob durch die Invivo-.NO-Gabe der arteriosklerotische Prozeß nicht eher beschleunigt wird.

Role for NADPH/NADH Oxidase in the Modulation of Vascular Tonea

Abstract: The endothelium modulates vascular tone by producing vasodilator andvasoconstrictor substances. Of these, the best characterized and potentially most important are nitric oxide (NO•) and O2−•. These small molecules exhibit opposing effects on vascular tone and chemically react with each other in a fashion that negates their individual effects and leads to the production of potentially toxic substances, such as peroxynitrite (ONOO−). These dynamic interactions may likely have important implications, altering not only tissue perfusion but also contributing to the process of atherosclerosis. The precise O2−• source within vascular tissue remains to be determined. Recent work demonstrated that in endothelial cells as well as in vascular smooth muscle cells, a membrane‐associated NAD(P)H‐dependent oxidase represents the most significant O2−• source. Interestingly, this oxidase is activated upon stimulation with angiotensin II, suggesting that under all conditions of an activated circulating and/or local renin‐angiotensin system endothelial dysfunction secondary to increased vascular O2−• production is expected.

New Insights Into Mechanisms UnderlyIng Nitrate tolerance

The hemodynamic and anti-ischemic efficacy of organic nitrates is rapidly blunted due to the development of nitrate tolerance. The mechanisms underlying this phenomenon remain poorly understood and likely involve several independent factors. More recent experimental observations suggest that tolerance may be the consequence of intrinsic abnormalities of the vasculature, including enhanced vascular superoxide and endothelin production. Superoxide anions degrade nitric oxide derived from nitroglycerin, whereas autocrine-produced endothelin within vascular smooth muscle sensitizes the vasculature to circulating neurohormones, such as catecholamines and angiotensin II, all of which may compromise the vasodilator potency of nitroglycerin. Interestingly, these vascular consequences of in vivo nitroglycerin treatment can be mimicked by incubating cultured endothelial and smooth muscle cells with angiotensin II. Further, nitrate tolerance and rebound following sudden cessation of prolonged nitroglycerin therapy can be prevented by concomitant treatment with high-dose angiotensin-converting enzyme inhibition or angiotensin-I receptor blockade. These data strongly suggest that increased circulating levels of angiotensin II, which are encountered during in vivo nitroglycerin treatment, initiate cellular events that ultimately attenuate the nitroglycerin vasodilator effects during prolonged treatment periods.

Adverse effects of nitroglycerin treatment on endothelial function, vascular nitrotyrosine levels and cGMP-dependent protein kinase activity in hyperlipidemic Watanabe rabbits.

OBJECTIVE With the present studies we sought to determine how treatment with nitroglycerin (NTG) affects endothelial function, oxidative stress and nitric oxide (NO)-downstream signaling in Watanabe heritable hyperlipidemic rabbits (WHHL). BACKGROUND In vitro experiments have demonstrated potent antiatherosclerotic effects of NO suggesting that treatment with NO-donors such as NTG could compensate for the diminished availability of endothelial NO. Nitric oxide may, however, not only be scavenged by reaction with endothelium-derived superoxide but also form the potent oxidant and inhibitor of vascular function, peroxynitrite (ONOO(-)). METHODS Watanabe heritable hyperlipidemic rabbits were treated for three days with NTG patches. Normolipidemic New Zealand White rabbits (NZWR) served as controls. Endothelial function was assessed ex vivo with organ chamber experiments and vascular superoxide was quantified using lucigenin (5 and 250 microM) and CLA-enhanced chemiluminescence. Vascular ONOO(-) formation was determined using nitrotyrosine antibodies. The activity of the cGMP-dependent kinase (cGK-I) was assessed by determining the phosphorylation of vasodilator-stimulated phosphoprotein VASP (P-VASP). RESULTS Nitroglycerin treatment caused endothelial dysfunction in NZWR and WHHL, associated with an increase in superoxide and ONOO(-) production and a substantial drop in cGK-I activity. In vivo NTG-treatment decreased lipophilic antioxidants (alpha- and beta-carotene) in NZWR and WHHL. Treatment of NZWR with NTG also decreased plasma extracellular superoxide dismutase (EC-SOD)-activity. CONCLUSIONS Nitroglycerin treatment of WHHL with exogenous NO worsens rather than improves endothelial dysfunction secondary to increased formation of superoxide and/or peroxynitrite leading to decreased cGK-I activity. The decrease in plasma levels of alpha- and beta-carotene may be at least in part due to a decrease in EC-SOD activity.

Platelet Glycoprotein IIb/IIIa Receptor Blockade Improves Vascular Nitric Oxide Bioavailability in Patients With Coronary Artery Disease

Background—Platelet glycoprotein IIb/IIIa receptor blockade not only enhances epicardial flow but also improves microvascular perfusion. Inhibition of abnormal platelet–endothelial interactions may contribute to this beneficial effect. The present study was designed to determine whether glycoprotein IIb/IIIa receptor blockade influences endothelial vasomotor function and NO bioactivity in patients with coronary artery disease. Methods and Results—Forty patients with symptomatic coronary artery stenosis were studied before planned percutaneous coronary intervention. By using venous occlusion plethysmography, endothelium-dependent and -independent vasodilation was determined by measuring forearm blood flow responses to acetylcholine with and without NG-monomethyl-l-arginine (L-NMMA) and sodium nitroprusside. Vascular function tests were repeated during glycoprotein IIb/IIIa receptor blockade by tirofiban in 27 patients and by eptifibatide in 13 patients. A subgroup of 10 patients was retested 6 hours after stopping infusion of tirofiban. Glycoprotein IIb/IIIa receptor blockade by both substances improved acetylcholine-induced vasodilation and L-NMMA responses. Six hours after withdrawal of tirofiban infusion, the beneficial effects were not evident. Sodium nitroprusside–induced vasodilation was not changed by glycoprotein IIb/IIIa receptor blockade. Conclusions—These findings support the concept that abnormal platelet-endothelial interactions contribute to endothelial dysfunction and impaired NO bioactivity in patients with symptomatic coronary artery disease.